Abstract: This study investigated the degradation efficiency and enzymatic characteristics of two fungal enzymes—GsLiP (lignin peroxidase derived from Gelatoporia subvermispora) and CrMnp (manganese peroxidase derived from Ceriporiopsis rivulosa)—against deoxynivalenol (DON). Under optimized conditions of pH4.5 and 30 ℃, both enzymes exhibited significant DON-degrading activity, achieving degradation rates exceeding 50% within 24 h. Molecular docking analysis revealed strong binding affinities between the enzymes and DON, with binding energies of −34.78 kcal/mol for GsLiP and −33.38 kcal/mol for CrMnp, supporting their high degradation potential. DON cytotoxicity was significantly reduced following enzymatic treatment, as evidenced by a cell inhibition rate of less than 10% in Caco-2 cells at a DON concentration of 1 μg/mL. In a wheat matrix, GsLiP and CrMnp achieved degradation efficiencies of 23.07%±0.59% and 24.63%±0.20%, respectively. These findings underscore that GsLiP and CrMnp can effectively degrade DON under mild conditions, significantly reducing its toxicity, and highlight their potential applications in mitigating DON contamination in food safety and agricultural contexts.